This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid molecules coding for an enzyme involved in pyrimidine biosynthesis in plants, especially in seeds.
Orotidine 5xe2x80x2-monophosphate decarboxylase (OMP decarboxylase) catalyzes the final reaction in pyrimidine nucleotide biosynthesis, converting OMP to uridine 5xe2x80x2-monophosphate (UMP). In eukaryotes, this enzyme also performs the next-to-last step of linking phosphoribosyl-pyrophosphate (PRPP) to orotate to form OMP (Reyes and Guganig (1975) J Biol Chem 250:5097-108; Traut et al. (1980) Biochemistry 19:6062-8). The enzyme is a target for feedback inhibition wherein UTP and UMP both reduce its activity. In prokaryotes, in contrast, there is no feedback inhibition, and and the last two enzymatic reactions are not coupled.
Nucleotides are required for the synthesis of DNA and RNA, and are indirectly responsible for protein synthesis, due to the requirement for ribosomes and tRNAs in translation. Therefore, pyrimidine biosynthesis is a key metabolic pathway in all eukaryotes. Manipulation of this pathway is neverthelss possible since mutations to key enzymes can be partially overcome by feeding cells having potentially lethal mutations with CTP, UTP, or their mono- or diphosphate derivatives. Inhibitors of OMP decarboxylase have been identified which vary in their efficacy among different organisms, implying that engineering of the active site may yield enzymes that are more or less sensitive to inhibition (Shostak and Jones (1992) Biochemistry 31:12155-61). It is believed that overexpression or inhibition of OMP decarboxylase in plants may be useful for enhancing growth rates, developing new herbicides, developing new fungicides, developing new insecticides, or selectively altering development of individual organs.
The present invention concerns an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a first nucleotide sequence encoding a polypeptide of at least 200 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, and 12, or (b) a second nucleotide sequence comprising the complement of the first nucleotide sequence.
In a second embodiment, it is preferred that the isolated polynucleotide of the claimed invention comprises a first nucleotide sequence which comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, and 11 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, and 12.
In a third embodiment, this invention concerns an isolated polynucleotide comprising a nucleotide sequence of at least one of 800 (preferably at least one of 500, most preferably at least one of 400) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, and 11 and the complement of such nucleotide sequences.
In a fourth embodiment, this invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to at least one suitable regulatory sequence.
In a fifth embodiment, the present invention concerns an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
In a sixth embodiment, the invention also relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
In a seventh embodiment, the invention concerns an OMP decarboxylase polypeptide of at least 200 amino acids comprising at least 85% identity based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, and 12.
In an eighth embodiment, the invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of an OMP decarboxylase polypeptide or enzyme activity in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; (c) measuring the level of the OMP decarboxylase polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and (d) comparing the level of the OMP decarboxylase polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level of the OMP decarboxylase polypeptide or enzyme activity in the host cell that does not contain the isolated polynucleotide.
In a ninth embodiment, the invention concerns a method of obtaining a nucleic acid fragment encoding a substantial portion of an OMP decarboxylase polypeptide, preferably a plant OMP decarboxylase polypeptide, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 800 (preferably at least one of 500, most preferably at least one of 400) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, and 11, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a substantial portion of an OMP decarboxylase amino acid sequence.
In a tenth embodiment, this invention relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding an OMP decarboxylase polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
In an eleventh embodiment, this invention concerns a composition, such as a hybridization mixture, comprising an isolated polynucleotide of the present invention.
In a twelfth embodiment, this invention concerns a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the chimeric gene of the present invention or an expression cassette of the present invention; and (b) growing the transformed host cell, preferably a plant cell, such as a monocot or a dicot, under conditions which allow expression of the OMP decarboxylase polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.
In a thirteenth embodiment, this invention relates to a method of altering the level of expression of an OMP decarboxylase in a host cell comprising: (a) transforming a host cell with a chimeric gene of the present invention; and (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of the OMP decarboxylase in the transformed host cell.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of an enzyme involved in primidine biosynthesis, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding an OMP decarboxylase polypeptide, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of an OMP decarboxylase in the transformed host cell; (c) optionally purifying the OMP decarboxylase polypeptide expressed by the transformed host cell; (d) treating the OMP decarboxylase polypeptide with a compound to be tested; and (e) comparing the activity of the OMP decarboxylase polypeptide that has been treated with a test compound to the activity of an untreated OMP decarboxylase polypeptide, thereby selecting compounds with potential for inhibitory activity.